Abstracts from the AIDS Vaccine 2011 Conference

Appended below is a highly subjective selection of highlights, including new data on the role of CD4 T cell responses in control of HIV, an intriguing study linking the nucleotide composition of lentiviruses to their pathogenicity, and results from animal modelers attempting to recapitulate both the recent RV144 vaccine trial in Thailand and the STEP trial of Merck’s now-discontinued HIV vaccine candidate. Links to e-Posters are included if they are available on the conference website.

Background: A combination vaccine based on the recombinant canarypox vector ALVAC-HIV and HIV gp120 envelope glycoprotein protected nearly one third of vaccinees from HIV acquisition but afforded no protection from CD4+ T-cell loss or high virus levels in vaccines that became infected in the RV144 trial in Thailand. This result was surprising, given the limited ability of either vaccine component to induce CD8+ T-cell responses or elicit broadly neutralizing antibodies. An animal model able to predict HIV vaccine efficacy for humans could hasten progress in our understanding of the immune responses that contribute to protection.

Methods: We vaccinated Indian rhesus macaques with an ALVAC-SIV and SIVgp120 immunization regimen that mimics the RV144 trial. At the end of the immunization regimen, the vaccinated animals received a titered mucosal challenge with a dose of SIVmac251 shown to transmit a limited number of variants to naive controls, recapitulating human mucosal HIV transmission.

Results: Vaccine immunogenicity and efficacy in macaques were similar to observations in humans; as in RV144, one third of vaccinated macaques were protected from acquisition SIVmac251. Viral load and CD4+ T cell numbers in animals that did become infected were not different from controls. Animals protected from infection had equivalent T-cell responses but higher avidity binding antibodies to gp120 compared to unprotected animals.

Conclusion: Titered intrarectal challenge of macaques with SIVmac251 shows potential to accurately model clinically observed vaccine efficacy, suggesting a role for this model in understanding protective responses elicited by HIV preventive vaccines. Consistent with the results from RV144, our findings suggest non- neutralizing antibody responses may play a role in protection from SIV/HIV acquisition.

Background: During acute HIV-1 infection, early control of vi- remia and establishment of viral set point have been widely attributed to HIV-specific CD8 T-cell responses. However, despite increasing evidence for direct antiviral activity by CD4 T-cells in other infections, the impact of HIV-specific CD4 T-cells on viral control has not been studied.

Methods: We studied 26 acutely infected, treatment-naive subjects and monitored their clinical outcome for up to 3 years post- infection. Baseline HIV-specific CD4 T-cell responses were assessed for degranulation (CD107a), IFNc secretion, expression of granzymes (GrzA,B,K) and perforin. HIV-specific cytolytic CD4 T-cell responses were investigated longitudinally in a subset of 11 subjects with similar peak viral loads for 1 year post-infection.

Results: Among the patients followed longitudinally, 6 progressed to a low viral set point, while 5 progressed to a significantly higher set point (134,020 vs. 11,234 copies/ml; p = 0.004). Interestingly, a significant expansion of HIV-specific cytolytic CD4 cell responses was observed in individuals who controlled viral replication compared to those who progressed to a high viral set point (IFNg: p = 0.038, CD107a: p = 0.042). Importantly, this expansion was observed early post-infection, prior to the divergence of viral load or CD4 T cell counts between the two groups. Examination of the baseline HIV-specific CD4 responses revealed a distinct GrzA-enriched cytolytic phenotype that was highly associated with subsequent viral control. Strikingly, Kaplan-Meier analysis of only the baseline cytolytic CD4 T-cell response in a larger cohort demonstrated that individuals with HIV-specific GrzA+ responses remained off HAART significantly longer than individuals with HIV-specific GrzA-responses (p = 0.0023).

Conclusion: Here we demonstrate that the rapid induction and expansion of a distinct cytolytic CD4 T-cell response during acute infection is significantly associated with viral control and disease outcome. These data suggest a pivotal role for HIV-specific cytolytic CD4 T-cells in the early control of viremia following acute infection and for future HIV vaccine design strategies.

P17.32 LB

Primary Immune Responses to Vaccinia Virus Vaccination: The Role of Cytotoxic Effector CD4+ T Cells in the Generation of Human T Cell Memory

Background: To understand the developmental stages of CD4+ T cell responses to viral infection and their differentiation into long-term memory cells, we studied individuals receiving primary vaccinia virus (VV) vaccination. This provides an attractive model for the study of human antiviral T cell responses as vaccination results in an acute infection that is cleared and leads to long-term protective immunity.

Conclusion: Generation of anti-viral human CD4+ T cell memory during primary immune responses is poorly understood. The role of CTL associated genes in this process has not been explored. Understanding their role in the generation of an effective memory may lead us closer to the development of a more effective HIV vaccine, such as enhancing the modestly efficacious HIV-1 vaccine used in the RV144 study.

Background: One of the foremost challenges in the development of an HIV-1 vaccine is that HIV-1 targets CD4-T cells, which are critically important in shaping the immune response to infection. It has been demonstrated that T follicular helper (TFH) CD4-T cells are pivotal for the induction of broadly neutralizing antibody (nAb) responses and for the generation of long-lived B-cell memory maturation. By cognate interaction and cytokine secretion, CD4-T cells expressing CD40L can induce class switching and somatic hypermutation in antigen specific B-cells, which is reflected in the expression of activation-induced cytidine deaminase (AID). Nonetheless, the role of TFH cells and their interaction with B-cells in HIV infection is currently unknown.

Methods: Ten subjects were identified during acute HIV infection and followed longitudinally over a median of three years. Neutralizing activity of plasma antibodies against a panel of present and past autologous and heterologous viruses was assessed using a single-replication cycle assay in which full-length envelope genes were incorporated into expression vectors (Monogram Biosciences). Phenotypic and functional characteristics of HIV-specific CD4-T cell responses and their impact on AID expression were assessed longitudinally by multiparameter flow cytometry.

Results: The breadth of the broadly neutralizing antibody response to heterologous HIV significantly increased over time in each patient. Interestingly, the expansion in heterologous breadth was not only significantly correlated with an increase in AID expression in B-cells, but also with CD4-T cell expression of PD-1, a marker that has been associated with TFH cells. Moreover, a positive association between AID expression and gp120-specific CD40L-responses was detected at the earliest time point.

Conclusion: Our data strongly indicate that the induction of broadly neutralizing antibody responses is linked to the function and activation of HIV-specific CD4-T cell responses. These findings will be pivotal in efforts to generate neutralizing antibody responses by vaccination strategies.

1Emory University, Atlanta, USA; 2Laboratory of Molecular Microbiology, National Institutes of Health, Bethesda, USA; 3Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, USA; 4School of Medicine, UT Health Science Center at San Antonio, San Antonio, USA; 5Department of Biostatistics, University of Pennsylvania, Philadelphia, USA; 6The AIDS and Cancer Virus Program, The National Cancer Institute, Frederick, USA

Background: The relative contribution of CD4+ T-cells to the control of virus replication during primary SIV infection rhesus macaques (RM) remains incompletely defined. To investigate the relationship between CD4+ T-cells and post-peak decline of viremia, we depleted CD4+ lymphocytes from non- inductive immunological sites of five RMs prior to SIV-infection.

Results: At the time of infection, CD4+ lymphocyte-depleted RM showed ten-fold lower CD4+ T cell levels in blood and LNs and normal CD4 + T-cell levels in RBs. After infection, CD4+ lymphocyte-depleted animals exhibited a similar peak of viremia but, in contrast to non-depleted RMs, showed no post-peak virus decline, and progressed more rapidly to AIDS. Importantly, no significant differences were found between depleted and undepleted RMs in terms of (i) availability of target cells; (ii) levels of SIV-specific CD8+ T cell responses; and (iii) SIV-specific humoral responses. Interestingly, peak viral load in depleted animals was associated with a striking increase in virus production by macrophages and dendritic cells.

Conclusion: These data indicate an essential role for CD4+ T-cells in mediating post-peak decline of viremia in SIV-infected RMs that may be associated with an ability to suppress virus production by macrophages. The complex balance between CD4+ T-cells as target cells and immune effectors may play a greater than anticipated role in the outcome of vaccine efficacy and should be considered during future vaccine design.

Background: The role of CD8+ T cells in containment of retroviral infections is well documented by studies of CD8+ T cell- mediated viral escape. It contrast, the immunological role of retrovirus-specific CD4+ T cells remains unclear, particularly in the setting of elite control. Here, we describe viral escape from CD4+ T cells concomitant with viral breakthrough and loss of elite control.

Methods: A SIVmac239-infected Indian rhesus macaque maintaining elite control of viremia (<1,000 vRNA copies/mL plasma) spontaneously lost control, experienced breakthrough viremia, and eventually succumbed to AIDS. We performed whole genome sequencing on plasma virus prior to the virus becoming undetectable and immediately following breakthrough and loss of elite control. We then investigated the T cell response directed against areas in the viral proteome exhibiting sequence divergence between the pre- and post-breakthrough time points.

Results: Comparison of the pre- and post-breakthrough viral sequences revealed surprisingly few amino acid substitutions post-breakthrough. These substitutions occurred within two previously defined and three previously undescribed CD8+ T cell epitopes, confirming the critical role of CD8+ T cells in control of viral replication. Interestingly, we identified two mutations within Gag, V63A and D205E, which occurred within CD4+ T cell epitopes. Both of these mutations abrogated CD4+ T cell recognition. While the V63A mutation also abrogated recognition of an overlapping CD8+ T cell epitope, the D205E mutation was solely targeted by a CD4+ T cell response. We confirmed that no CD8+ T cell responses were targeting this region. Finally, we demonstrated that viruses bearing the GagD205E mutation escaped CD4+ T cell effector function in vitro.

Conclusion: Virus-specific CD4+ T cells play an active role in the containment of retroviruses and are likely essential for the maintenance of low-level viremia. How these CD4+ T cells persist and exert their anti-viral function during retroviral infection remains unclear and warrants further investigation.

Conclusion: Lytic granule loading of CD8+ T cells and efficient delivery of active GrB to SIV-infected targets are associated with control of SIV infection in rhesus macaques, consistent with observations of HIV infection in humans. These findings suggest ICE is a correlate of control of viral replication in chronic SIV infection. They also suggest the use of cytotoxic capacity as a predictor of immunologic control in the vaccine setting should be tested.

OA09.02

ADCC Titers to HIV-Infected Cells Are Detectable in the Majority of Vaccine Recipients in the RV144 Trial

Background: A modest reduction in the rate of HIV-1 infection was observed among vaccine recipients in the RV144 trial who received a prime-boost vaccine regimen with recombinant canarypox and soluble gp120. Whereas virus-specific CD8+ T cell responses were largely undetectable, antibodies capable of binding to gp120 were detectable in nearly all vaccinated subjects. However, these antibodies were unable to neutralize primary HIV-1 isolates, suggesting that antibodies may have contributed to protection by non-neutralizing effector mechanisms.

Methods: To determine if vaccinated subjects made antibodies capable of directing antibody-dependent cell-mediated cytotoxicity (ADCC), we used a novel assay to measure ADCC titers against HIV-infected cells in pilot samples from the RV144 trial. This assay is based on the killing of an HIV-infected CD4+ T cell line by a CD16+ NK cell line in the presence of serial dilutions of plasma, thus allowing ADCC titers to be measured against virus infected cells expressing the native, oligomeric conformation of the HIV-1 envelope glycoprotein. Using this approach, ADCC titers in blinded plasma samples from 80 vaccine recipients and 20 placebo controls were measured at baseline (week 0) and at two weeks after the last boost (week 26) against target cells infected with the HIV-1 subtype AE isolate 92TH023.

Background: Genome-wide transcriptional studies in non-human primates have suggested that the quality and magnitude of early immune responses to SIV infection are associated with downstream disease progression. The aim of this study is to identify, monitor, and interpret the whole blood transcriptional signature of acute HIV-1 infection (AHI).

Methods: A total of 56 AHI patients and matching healthy controls from both Africa and the United States were distributed into training, test, and validation datasets. Whole blood from these subjects was obtained for transcriptional profiling using microarrays. Both gene and modular transcriptional framework analyses were utilized to interpret the signature of AHI and for comparisons with other disease signatures including: influenza, tuberculosis, sepsis, and chronic HIV following interruption of anti-retroviral therapy (ART). Additional samples were collected at 1, 2, 4, 12, and 24 weeks post-enrollment for training and test set AHI subjects, to establish the longevity of the signature in the presence or absence therapy.

Results: We identified a robust AHI signature with increased activity in: interferon, cell cycle, cytotoxic, plasma cell, and B-cell modules amongst others. This activity was unique when compared to signatures obtained from patients with other infections. Only interferon and cell cycle activity was observed in both AHI subjects and chronically infected patients following interruption of therapy. Notably, 20% of AHI patients were observed to express a quiescent signature that clustered independent of the highly active signature described above. We found that these patients had significantly lower viral set-points (p = 0.01) when compared to patients with the active signature. Finally, longitudinal analysis demonstrated that the active AHI signature can persist independent of viral load in ART treated patients and up to 6 months in the absence of therapy.

Conclusion: Transcriptional monitoring of early HIV infection offers both quantitative and qualitative assessments patient responses that may be predictive of long-term disease progression.

P09.12

Correlation Between AIDS Pathology and the Biased Nucleotide Composition of Lentiviruses

Background: AIDS results from generalized immune activation after years of chronic HIV replication. However, most African primate species naturally infected with lentiviruses remain healthy. Addressing this issue is crucial for understanding AIDS pathogenesis. Because the genomes of lentiviruses present a particularly biased nucleotide composition compared to that of their primate hosts, we wondered whether host recognition of biased viral nucleic acids could induce hyper-responsiveness to HIV and AIDS pathogenesis.

Methods: We compared the nucleotide composition (A/C/G/T frequencies) of pathogenic and non-pathogenic primate lentiviruses with that of their hosts for every infection described in the literature. We measured the nucleotide divergence by computing the Chi-2 distance between the A/C/G/T frequencies in the complete viral sequence and the coding sequences of each host organism.

Results: We found that primate lentiviruses having the most divergent nucleotide composition compared to their hosts induce AIDS, whereas less divergent lentiviruses cause non-pathogenic infections. Similarly, the relative pathogenicity of HIV-1 subtypes correlates with their nucleotide divergence to the human genome.

To understand this observation at a molecular level, we investigated the ability of HIV-1 RNA fragments to stimulate in vitro the synthesis of type I interferon (IFN-I). We found that the nucleotide divergence of RNA fragments strongly correlates with type-I IFN activity. Based on these observations, we designed a large-scale, nucleotide-optimized SIV sequence derived from the pathogenic clone SIVmac239. We produced the corresponding artificial virus in lymphocyte cell line and analysed its capacity to induce type-I IFN in different cellular models. This synthetic virus presented the same replicative capacity than WT virus and a reduced ability to induce type-I IFN.

Conclusion: Overall, these data suggest for the first time a direct link between the nucleotide composition of lentiviruses and their pathogenicity. They also describe the synthesis a novel artificial SIV harbouring an attenuated pathogenic potential.

Background: Due to the current dual-epidemic of HIV+/AIDS and TB, functional understanding of HIV+/MTB co-infection immunology is critical for furthering vaccine and therapeutic strategies against both pathogens. IL-10 is an anti-inflammatory cytokine, produced by lymphocytes and monocytes, that has been shown to down-regulate expression of anti-viral and anti-bacterial cytokines such as IFN-gamma, IL2 and TNF-alpha. Elevated IL-10 levels have been reported during chronic human HIV infection and ex vivo IL-10 receptor blockade in the same patients has been shown to increase HIV specific T cell function.

Conclusion: This study concluded that there is a synergistic upregulation of IL-10 in HIV+/TB active patients as compared to HIV and MTB mono-infection. Additionally, this study concludes that novel ex vivo enhancement of MTB and HIV specific CD4 T cell function is possible with IL-10Ra blockade in HIV+/LTBI subjects.

P17.06

Cryptic Epitopes from Alternative Reading Frames Restricted by HLAs Associated with a Good Prognosis Are Frequently Recognized in HIV Infection

Background: Cryptic epitopes (CE) are products of translation of alternate reading frames (ARF, 2 sense and 3 antisense) that are commonly targeted during HIV/SIV infection. To understand the full extent of contributions of HIV specific CE in HIV-1 pathogenesis, we performed mapping of the CE derived from ARFs of nine HIV-1 encoded proteins.

Results: We predicted 520 (B*27), 955 (B*57) and 1027 (B*5801) potential CE in each of the five ARFs of the nine HIV-1 encoded proteins. The antisense frames encoded a majority of these CE. Predicted peptides with >50% probability of being an epitope were synthesized; B*27 (N = 30), B*57 (N = 39) and B*58 (N = 90). Overall, 26% and 2% of CHI patients and seronegative donors respectively had CE responses (p = 0.0006, Fischer’s exact). The overall responder frequency for HLA specific CE response was 22% (range 6%-43%) with the highest recognition frequency noted for HLA-B*5801/B*57 patients (p = 0.005, Fischer’s exact). In our comprehensive mapping analysis, 17% patients targeted CE from gag/pol ARFs and all responders were either HLA-B*27, B*57 or B*5801. Among these, the most frequently targeted were pol 2 (31%) followed by gag 2 and 5 (25%) frames.

Conclusion: These data underscore the importance of CE targeting especially those that are presented by the so-called ‘‘protective alleles’’ in HIV-1 infection and hence have implications for vaccine design.

Background: Although HIV-specific CD4+ T-cells are preferentially infected, there is growing evidence that these cells play a pivotal role in control of viremia. However, little is known about the recognition and HLA-class II restriction of HIV-specific CD4+ T-cells in the setting of chronic or spontaneously controlled HIV-infection. Yet, this knowledge will be crucial for the induction of protective immunity in a prophylactic vaccine.

Results: HIV-specific CD4+ T-cell responses were detected in all patient subgroup yet the breadth of these responses was significantly expanded in HIV-controllers (p = 0.029). Most HIV- specific CD4+ T-cells targeted epitopes within Gag, Nef and gp120. Strikingly, we observed significant differences in the immunodominance profile between patient subgroups that distinguished not only elite controllers from rapid progressors, but also from viremic controllers. While elite controllers dominantly targeted a tight cluster of conserved epitopes within p24, chronic progressors preferentially targeted epitopes within the C1/C3 domain of gp120. Moreover, the ratio of Env- and Gag-specific responses was a clear indicator of viral control. A multivariate bootstrap analysis identified four distinct Gag epitopes that were associated with spontaneous control (Probability = 0.60–0.85), while a single gp120 peptide in C3 was associated with high viremia (Probability = 0.82). Detailed characterization showed that promiscuous binding to multiple HLA-DR alleles occurs frequently, and revealed that HLA-DRB1*0701 is associated with slow disease progression (Probability = 0.88).

Conclusion: Our data demonstrate that significant differences exist in protein targeting by HIV-specific CD4+ T-cells between controllers and progressors. We also identified distinct epitopes associated with viral control that are conserved and bind promiscuously to multiple HLA-class II alleles, which will be important for vaccine design.

Methods: To develop an animal model to understand these results we determined if rhesus macaques chronically infected with a host range mutant adenovirus type-5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag-Pol- Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques during a series of repeated penile SIVmac251 exposures.

Results: We found that 2 of 9 rhesus macaques infected with Ad5hr prior to immunization became SIV infected after penile exposure to 103 TCID50 of SIVmac251. In contrast none of the 34 unimmunized animals, including 8 Ad5 seropositive animals immunized with the empty vector; Ad5 seronegative animals immunized with the Ad5 SIV vaccine (n = 9) or empty vector (n = 9) and 8 naive control animals became SIV infected after penile exposure to the same dose of SIVmac251. At the lowest SIV exposure dose (103 TCID50), the risk of infection was greater for animals with Ad5 titers at the time they were immunized with the MRKAd5 SIVmac239 subtype Gag-Pol-Nef vaccine compared to all the other animal groups (p = 0.004 based on log rank test). Penile exposure to more concentrated virus inoculums produced similar rates of infection in all animals groups. The Ad5 SIV vaccine induced CD8+ T cell responses in approximately 70% monkeys, which is similar to the proportion of humans that responded to HIV-1 vaccine.

Conclusion: The results of the NHP study described here recapitulate the results of the STEP trial and demonstrate that the results of NHP studies using challenge viruses and routes of exposure designed to mimic the variety and complexity of HIV-1 sexual transmission can reflect the outcomes of AIDS vaccine efficacy trials.

Background: The genetic diversity of HIV antigens represents a paramount challenge in the development of vaccines. Here, we establish rationale for circumventing this obstacle by eliciting cellular immune responses against stable human endogenous retrovirus K (HERV-K) antigens. Protein-level expression of HERV-K antigens has not been observed in healthy tissues, but does occur in various malignancies. We hypothesized that HIV would disrupt control of HERV-K expression within infected cells, targeting them for elimination by HERV-K-specific CD8+ T-cells.

Methods: HERV-K expression was assessed by qPCR, western blot, immunohistochemistry and mass-spectrometry. HERV-K- specific T-cell responses were identified by ELISPOT and a HERV-K-Env-specific CD8+ T-cell was cloned by IFN-g capture. Additional HERV-K-specific T-cell lines and clones were obtained by a dendritic-cell based expansion method. Recognition and elimination of HIV-infected cells were assessed by flow cytometry.

Results: HIV-infection resulted in HERV-K Gag and Env protein expression in primary CD4+ T-cells. HERV-K Gag and Env specific CD8+ T-cells specifically responded to, and eliminated, HIV-infected cells in an MHC-I-restricted manner. This recognition was dependent upon Vif, however expression of Vif alone was insufficient to recapitulate a response. A HERV-K-Env-specific CD8+ T-cell clone displayed comprehensive elimination of cells infected with a panel of 23 diverse HIV-1 and HIV-2 primary isolates as well as with SIVmac251.

Conclusion: The de-repression of HERV-K expression in HIV-infected cells, for which Vif is necessary but insufficient, constitutes a marker of infection that can be targeted by HERV-K-specific CD8+ T-cells. The unprecedented breadth of reactivity of HERV-K-specific T-cell responses against diverse lentiviruses, both implied by the proposed mode of action and observed in the current study, comprises an enticing advantage over HIV-1-specific T-cell responses which could be exploited in the development of HERV-K-targeted vaccines to treat or prevent HIV infection. These data also provides rationale for considering a role for HERV-K-specific T-cell responses in natural control of HIV.

P18.26 LB

Immunological Correlates of Protection Against Acquisition of HIV-1 Infection in the iPrEx Trial

Background: Pre-exposure prophylaxis for HIV-1 prevention in men who have sex with men (MSM) has been shown to reduce overall HIV-1 incidence by 44%. We hypothesized that exposure to HIV-1 before enrollment could have stimulated cell mediated immunity, which contributed to the observed protection.

Methods: We studied immune responses in highly exposed MSM enrolled in the iPrEx study who either seroconverted (SC; N = 93) or remained seronegative throughout follow up for up to 132 weeks (ESN; N = 485). Pre-infection time-points from SC participants were matched with time-points from ESN controls and analyzed for HIV-1 specific T-cell responses to p24, Nef, Vif, integrase (Int), reverse transcriptase (RT), and protease (Prot) using a standard IFNg ELIspot assay. Non-parametric tests were used to assess statistical significance of the comparisons between responses in SC and ESN participants.